Human tendon collagen: Aging and crosslinking

The level ofcrosslinking in human tendon collagen ofvarious ages was assessed by measuring the amount of solubilized peptides after cyanogen bromide digestion. The result suggests that progressive crosslinking occurs throughout adult life. The fluorescence properties of the aged collagen indicate that nonenzymatic browning or the Maillard reaction may be involved in the crosslinking process. There is a common presumption that progressive crosslinking occurs in collagen throughout life. Accumulation of crosslinks is thought to make the organs stiff and to impair their function. However, recent studies on the aging of bovine tendon collagen refuted this presumption (1, 5). In these studies, the level of crosslinking was assessed by analyzing the peptides released by cyanogen bromide digestion. The results showed that age-related crosslinking was completed by the time the animal matures and there is no evidence for any subsequent, progressive crosslinking with age after maturity (l, 5). In contrast, Kohn reported that duramater collagen from human adult was solubilized incompletely by the treatment with cyanogen bromide and that there was an age-dependent decrease in the amount of solubilized peptides (4). The result supports progressive crosslinking occurring throughout life. The discrepancy may be ascribed either to the difference in animal species or in tissues. This communication deals with the aging of human tendon collagen. The solubility by cyanogen bromide digestion of human tendon collagen was found to decrease markedly with age, indicating that progressive crosslinking occurs throughout human adult life. The fluorescence properties of the aged collagen suggests that nonenzymatic browning or the Maillard reaction may be involved in the crosslinking process. Samples of human Achilles tendon were obtained at autopsy. They were cleaned ofadhering tissues, minced, soaked in 0.5 M NaCl at 4°C for 24 h, washed with cold water, dehydrated in acetone and dried under vacuum. Each sample (20 mg) was digested with cyanogen bromide (50 mg) in 70% formic acid (2 ml) at 20°C for 3 h. After digestion each sample was diluted 10-fold in water and centrifuged at 10,000 g for 20 min. The supernatant was evaporated under reduced pressure and the dried sample was hydrolyzed with 6M HCI (2 ml) at 110°C for 24 h. The precipitate was also hydrolyzed in the same manner. Hydroxyproline in the hydrolysate was determined (9) and the amount of collagen was estimated. In order to measure fluorescent properties, the collagen sample was solubilized as follows. Each sample (20 mg) was suspended in 50 mM Tris-5 mM CaCl2, pH 7.5 (2 ml) and heated at 65°C for 15 min. After cooling, pronase E (2 mg; Kaken Kagaku) was added and the mixture was incubated at 37°C for 1 h. A small amount of insoluble material was removed by centrifugation. Fluorescence spectrum of the supernatant solution was measured in a Hitachi 204 fluorescnce spectrophotometer. As shown in Table l, the collagen from young subject was completely solubilized by the cyanogen bromide treatment, whereas the collagen from aged subject was markedly resistant to the solubilization by cyanogen bromide. There was an age-dependent decrease in the amount of solubilized peptides. This result is similar to that obtained for human dura mater collagen (4) and different from that for bovine tendon collagen (1, 5). Pretreatment of the 79 yeard-old collagen with i